Endoluminal local delivery of PCNA/cdc2 antisense oligonucleotides by porous balloon catheter does not affect neointima formation or vessel size in the pig coronary artery model of postangioplasty restenosis

Localized delivery of antisense oligonucleotides directed against cell cycle regulatory proteins has been proposed as a means to prevent restenosis after angioplasty. To test whether single endoluminal delivery of a combination of proliferating cell nuclear antigen (PCNA) and cell-division cycle 2 kinase (cdc2) antisense might affect restenosis, we delivered 2 ml of lipid-complexed PCNA/cdc2 antisense oligomers (1.35 mg) to the coronary arteries of pigs after balloon overstretch angioplasty (AS group) and performed planimetric histomorphometry on arterial sections of the tissue, harvested at 4 wk. Compared with controls receiving 3′–5′ reversed sequence oligomers (REV group), there were no differences in absolute intimal area (AS 1.36 ± 0.08 mm², REV 1.23 ± 0.10 mm², P = NS), intimal area normalized to extent of injury (AS 0.67 ± 0.03, REV 0.77 ± 0.10, P = NS), or vessel perimeter (AS 7.72 ± 0.19 mm, REV 7.36 ± 0.22 mm, P = NS). We conclude that single endoluminal delivery of antisense against key cell cycle regulatory proteins does not affect neointima formation or vessel size in this model of restenosis. Cathet. Cardiovasc. Diagn. 41:348–353, 1997. © 1997 Wiley-Liss, Inc.     

Selective changes in hippocampal neuropeptide Y neurons following removal of the cholinergic septal inputs

The number and distribution of subpopulations of hilar interneurons containing neuropeptide Y (NPY), somatostatin (SOM), or γ-aminobutyric acid (GABA) immunoreactivities were examined in the hilus of the dentate gyrus following removal of the cholinergic septal inputs. One, 2, 4, 8, 12, and 24 weeks after intracerebroventricular injections of immunotoxin, consisting of antibody to the low-affinity nerve growth factor receptor conjugated to saporin (192 IgG-saporin), lesioned rats were processed simultaneously with controls for NPY, SOM, or GABA immunolabeling. Across all time points, the number of NPY-labeled neurons was reduced to a statistically significant level (paired t-test, P = 0.001) in the injected rats (73% of control values, on average). The decrease in the number of NPY-labeled neurons was not limited to any particular subregion rostrally but appeared greater in the central region caudally. The size of NPY-labeled neurons did not differ statistically between control and immunolesioned rats examined at 1, 2, and 24 week time points. In contrast, the number of both SOM- and GABA-immunoreactive neurons in injected rats did not appear to be affected in any consistent manner. Examination of the hilus in adjacent Nissl-stained sections with the optical dissector revealed that although the total number of small nonprincipal cells (5–15 μm in diameter) did not appear affected at the 4-week time point, there was a statistically significant (P = 0.03) reduction across the 8–24-week time points (to 80% of control values, on average). Dual-labeling studies on separate rats showed that a small subpopulation of the NPY- and SOM-labeled neurons, primarily in the infragranular hilus, were colocalized with neurons containing GABA immunoreactivity (18% and 5%, respectively). These studies demonstrate that removal of the cholinergic septal inputs (1) can cause relatively rapid, selective decreases in the number of NPY-immunoreactive hippocampal interneurons and (2) appears to lead to the death of hippocampal interneurons over a longer time course. The changes in NPY immunoreactivity seem to occur in the portion of interneurons that probably does not contain either SOM or GABA immunoreactivity. J. Comp. Neurol. 386:46–59, 1997. © 1997 Wiley-Liss, Inc.     

Hexavalent sperm-binding IgG antibody released from vaginal film for development of potent on-demand nonhormonal female contraception

Nonhormonal products for on-demand contraception are a global health technology gap; this unmet need motivated us to pursue the use of sperm-binding monoclonal antibodies to enable effective on-demand contraception. Here, using the cGMP-compliant Nicotiana-expression system, we produced an ultrapotent sperm-binding IgG antibody possessing 6 Fab arms per molecule that bind a well-established contraceptive antigen target, CD52g. We term this hexavalent antibody “Fab-IgG-Fab” (FIF). The Nicotiana-produced FIF had at least 10-fold greater sperm-agglutination potency and kinetics than the parent IgG, while preserving Fc-mediated trapping of individual spermatozoa in mucus. We formulated the Nicotiana-produced FIF into a polyvinyl alcohol–based water-soluble contraceptive film and evaluated its potency in reducing progressively motile sperm in the sheep vagina. Two minutes after vaginal instillation of human semen, no progressively motile sperm were recovered from the vaginas of sheep receiving FIF Film. Our work supports the potential of multivalent contraceptive antibodies to provide safe, effective, on-demand nonhormonal contraception.

Despite the availability of potent and low-cost, long-acting, reversible contraceptives, many women continue to use on-demand contraceptives due to infrequent sexual activity. In addition, many women strongly prefer nonhormonal contraceptives because of the real and/or perceived side effects associated with existing hormonal methods (1–3). Indeed, the FDA-approved Vaginal Contraceptive Film (VCF) meets the contraceptive needs of many women as it provides a contraceptive method that is women-controlled, inexpensive, nonhormonal, discreet, and readily available over the counter. Unfortunately, VCF and most other spermicides use nonoxynol-9 (N9) as an active ingredient. N9 can damage the mucosal surfaces by disrupting the vulvar, vaginal, and cervical epithelium and substantially increases the risks of sexually transmitted infections (4–6). We believe there is a substantial unmet need for alternatives that can offer effective on-demand contraception and are free of exogenous hormones or detergents.Antisperm antibodies (ASA) to surface antigens on sperm (7) represent a promising class of molecules that could enable safe, on-demand, nonhormonal contraception. ASAs found in the vaginal secretions of some immune infertile women could prevent fertilization by stopping sperm from reaching the egg via two distinct mechanisms (8). First, ASAs can agglutinate multiple motile sperm into clumps that stop forward progression (9, 10). This mechanism is most effective at high sperm concentrations and is more potent with polyvalent antibodies (Abs) such as IgM. Second, ASAs can trap individual spermatozoa in mucus by forming multiple low-affinity Fc-mucin bonds between sperm-bound ASA and mucin fibers (11), resulting in individual sperm that simply shake in place, unable to assume progressive motility needed to reach the upper reproductive tract. Over time, sperm that are agglutinated or immobilized in mucus either die or are eliminated from the female reproductive tract (FRT) by natural mucus clearance mechanisms.Years ago, the discovery of the contraceptive potential of ASAs motivated the development of contraceptive vaccines. ASAs elicited by vaccination with sperm antigens offered considerable contraceptive efficacy, but this approach stalled due to unresolved variability in the intensity and duration of the vaccine responses in humans, as well as concerns that active vaccination might lead to irreversible infertility (12–14). In contrast, topical delivery of pharmacologically active doses of ASA in the vagina can overcome many of the key drawbacks of contraceptive vaccines by providing consistent amounts of Abs needed without risks of inducing immunity to sperm, thus making possible both consistently effective contraception and rapid reversibility. In good agreement with this concept, vaginal delivery of a highly multivalent antisperm IgM reduced embryo formation by 95% in a highly fertile rabbit model (15).This approach of topical passive immunocontraception has not been reported in humans, due in part to manufacturing and purification challenges with polyvalent Abs such as sIgA and IgM and the lower agglutinating potencies of IgG. To overcome these challenges, we report here a highly multivalent IgG that possesses 6 Fabs per IgG molecule, with Fab domains interspersed by flexible glycine-serine linkers arranged in a Fab-IgG-Fab orientation; we term this molecule FIF (Fig. 1A). To determine whether FIF may be useful for on-demand contraception, we produced FIF using a cGMP-compliant Nicotiana benthamiana manufacturing platform and formulated the FIF into a dissolvable vaginal film comprised of polyvinyl alcohol (PVA). We report here the in vitro characterization and in vivo potency of this vaginal FIF Film.Open in a separate windowFig. 1.Production of FIF-N-Film. (A) Schematic diagrams of antisperm FIF. The additional Fab is linked to the N terminal and C terminal of parent IgG using flexible glycine-serine linkers to assemble FIF. (B) SDS-PAGE analysis of FIF-N in native (nonreducing) and reducing conditions. Nonreducing SDS-PAGE showcases the total molecular weight of the Ab and reducing SDS-PAGE displays the molecular weight of the individual HC and LC of Ab. (C) Demonstration of the homogeneity of FIF-N after protein A and CHT chromatography using HPLC-SEC analysis. y-axis indicates the total percentage of Abs representing their theoretical molecular weights. (D) Image of water-soluble PVA film comprising of Nicotiana-produced FIF Ab.     

The pain behavior check list (PBCL): Psychometric properties in a college sample

The Pain Behavior Check List (PBCL) was designed to assess the frequency of four dimensions of pain behavior: distorted ambulation, affective distress, facial/audible expressions, and seeking help. This study evaluated theoretical factor structure, internal consistency, and construct validity of the PBCL in a nonclinical college sample. Results provided support for the four-factor oblique model, compared to the one-factor and the four-factor orthogonal models. The PBCL total and subscales showed satisfactory internal consistency. Support for convergent validity was demonstrated by high correlations between the PBCL and several measures of pain behavior and with other pain indices. In addition, results of the confirmatory factor analyses suggested that self-report measures of pain can be differentiated from self-report measures of anxiety and depression.     

Bcl-2 delays macrophage engulfment of human B cells induced to undergo apoptosis

The capacity to be recognized and engulfed by phagocytes is an important characteristic of cells dying by apoptosis. Phagocytosis of apoptotic cells occurs rapidly in vivo, probably prior to plasma membrane breakdown. While the molecular mechanisms mediating phagocytosis of apoptotic cells are beginning to be defined, little is yet known of the relationship between the cell-death program itself and the surface changes on the dying cells that signal for engulfment. Here, we investigate to what extent the apoptosis repressor Bcl-2 can modulate the recognition and phagocytosis of human B cells exposed to triggers of apoptosis. Burkitt lymphoma (BL)-derived, Bcl-2⁻ B cells were induced into apoptosis either by the Ca²⁺-ionophore ionomycin or by the inhibitor of protein synthesis cycloheximide. Apoptotic BL cells, but not viable BL cells, were recognized and phagocytosed by monocyte-derived macrophages. bcl-2-transfected BL populations showed a reduced capacity both to undergo apoptosis in response to these inducing agents and to interact with macrophages. Like their Bcl-2⁻ counterparts, Bcl-2⁺ BL cells interacted with macrophages only after activation of their apoptotic program as assessed by changes in nuclear morphology. These results demonstrate not only that continued protein synthesis in B cells undergoing apoptosis is not essential for their recognition by macrophages, but also that macrophage recognition of apoptotic B cells cannot be uncoupled from the cell-death program that is controlled by Bcl-2. In this respect, the behavior of B cells contrasts markedly with that of neutrophils in which Bcl-2 has been reported to inhibit apoptosis without affecting phagocytic clearance (Lagasse and Weissman, J. Exp. Med. 1994. 179: 1047).     

Comparison of two inspiratory: expiratory ratios during high frequency oscillation

The aim of this study was to compare gas exchange and volume delivery during high frequency oscillation at two frequently used inspiratory:expiratory (I:E) ratios: 1:2 and 1:1, other oscillatory settings being kept constant. A group of 13 infants with respiratory distress syndrome, median gestational age 28 weeks (range 23–36) and postnatal age 1 day (range 1–8) were studied. At the I:E ratio of 1:1 compared to 1:2 the median paCO₂ was lower, P < 0.05 (30 mmHg, range 22–47 vs 34 mmHg, range 27–46) and the volume delivered higher, P < 0.01 (2.6 ml/kg, range 1.2–5.6 vs 2.0 ml/kg, range 1.0–3.9). There was no significant difference in oxygenation levels at the two I:E ratios. In a related in vitro study, changing the I:E ratio from 1:2 to 1:1 increased the mean airway pressure by a median of 8.6% (range 2.9–28.1%). Conclusion Routinely maintained longer expiratory than inspiratory times during high frequency oscillation should be discouraged. Received: 6 November 1998 / Accepted: 30 March 1999     

Randomised controlled trial of budesonide for the prevention of post-bronchiolitis wheezing.

BACKGROUND: Previous studies suggest that recurrent episodes of coughing and wheezing occur in up to 75% of infants after acute viral bronchiolitis. AIM: To assess the efficacy of budesonide given by means of a metered dose inhaler, spacer, and face mask in reducing the incidence of coughing and wheezing episodes up to 12 months after acute viral bronchiolitis. METHODS: Children under the age of 12 months admitted to hospital with acute viral bronchiolitis were randomised to receive either budesonide or placebo (200 microg or one puff twice daily) for the next eight weeks. Parents kept a diary card record of all episodes of coughing and wheezing over the next 12 months. RESULTS: Full follow up data were collected for 49 infants. There were no significant differences between the two study groups for the number of infants with symptom episodes up to six months after hospital discharge. At 12 months, 21 infants in the budesonide group had symptom episodes compared with 12 of 24 in the placebo group. The median number of symptom episodes was 2 (range, 0-13) in those who received budesonide and 1 (range, 0-11) in those who received placebo. Because there is no pharmacological explanation for these results, they are likely to be caused by a type 1 error, possibly exacerbated by there being more boys in the treatment group. CONCLUSION: Routine administration of budesonide by means of a metered dose inhaler, spacer, and face mask system immediately after acute viral bronchiolitis cannot be recommended.     

Loss of microRNA-21 leads to profound stromal remodeling and short survival in K-Ras-driven mouse models of pancreatic cancer

The microenvironment of pancreatic cancer adenocarcinoma (PDAC) is highly desmoplastic with distinct tumor-restraining and tumor-promoting fibroblast subpopulations. Re-education rather than indiscriminate elimination of these fibroblasts has emerged as a new strategy for combination therapy. Here, we studied the effects of global loss of profibrotic noncoding regulatory microRNA-21 (miR-21) in K-Ras-driven p53-deleted genetically engineered mouse models of PDAC. Strikingly, loss of miR-21 accelerated tumor initiation via mucinous cystic neoplastic lesions and progression to locally advanced invasive carcinoma from which animals precipitously succumbed at an early age. The absence of tumor-restraining myofibroblasts and a massive infiltrate of immune cells were salient phenotypic features of global miR-21 loss. Stromal miR-21 activity was required for induction of tumor-restraining myofibroblasts in in vivo isograft transplantation experiments. Low miR-21 expression negatively correlated with a fibroblast gene expression signature and positively with an immune cell gene expression signature in The Cancer Genome Atlas PDAC data set (n = 156) mirroring findings in the mouse models. Our results exposed an overall tumor-suppressive function of miR-21 in in vivo PDAC models. These results have important clinical implications for anti-miR-21-based inhibitory therapeutic approaches under consideration for PDAC and other cancer types. Mechanistic dissection of the cell-intrinsic role of miR-21 in cancer-associated fibroblasts and other cell types will be needed to inform best strategies for pharmacological modulation of miR-21 activity to remodel the tumor microenvironment and enhance treatment response in PDAC.